LYVE1 Human 25-235 a.a.

What is LYVE1 and what is the significance of the 25-235 amino acid region?

LYVE1 (Lymphatic Vessel Endothelial Hyaluronan Receptor 1) is a type I integral membrane glycoprotein that functions as a receptor for hyaluronic acid (HA). The 25-235 amino acid region encompasses the extracellular domain containing the Link module, which is the prototypic HA binding domain of the Link protein superfamily. This specific region is critical for the protein's functionality as it contains the complete HA-binding domain while excluding the transmembrane and cytoplasmic portions of the full protein. The recombinant version of this fragment is particularly valuable for research as it provides the functional binding domain in a soluble form that can be used for various in vitro assays and structural studies .

How does LYVE1 Human 25-235 a.a. compare structurally to the complete LYVE1 protein?

The complete human LYVE1 protein consists of 322 amino acid residues forming a type I integral membrane polypeptide with a 212-residue extracellular domain, a transmembrane domain, and a cytoplasmic tail. The 25-235 a.a. fragment specifically represents the functional extracellular portion of the protein containing:

• The complete Link module (the HA-binding domain)

• N-terminal flanking sequences necessary for proper folding

• C-terminal sequences preceding the transmembrane domain

This truncated version maintains the full HA-binding capability while being soluble and easier to produce and manipulate experimentally. Comparative structural analysis shows that this fragment shares approximately 41% similarity with the CD44 HA receptor, particularly in the Link module region .

What are the known binding partners and interactions of LYVE1?

LYVE1 primarily interacts with hyaluronic acid (HA), an abundant extracellular matrix glycosaminoglycan. The protein can bind to both soluble and immobilized HA through its Link module domain. Beyond its primary ligand, LYVE1 has been demonstrated to:

• Function as a homodimer in its native state

• Interact with PDGFB (Platelet-Derived Growth Factor B)

• Bind to IGFBP3 (Insulin-like Growth Factor Binding Protein 3)

These protein-protein interactions suggest broader signaling capabilities beyond simple HA binding. The extracellular domain (25-235 a.a.) is responsible for most of these interactions, making this fragment particularly valuable for interaction studies and ligand screening approaches .

What is the tissue distribution pattern of LYVE1, and are there recent revisions to this understanding?

While traditionally considered a specific marker for lymphatic endothelium, recent research has revealed a more complex expression pattern for LYVE1. Current understanding indicates:

• Primary expression in endothelial cells lining lymphatic vessels

• Expression in normal hepatic blood sinusoidal endothelial cells in both humans and mice

• Absence or reduced expression in angiogenic blood vessels of liver tumors

• Weak expression in the microcirculation of regenerative hepatic nodules in cirrhosis

This expanded expression profile challenges the previous paradigm of LYVE1 as an exclusive lymphatic marker. This discovery has significant implications for researchers using LYVE1 as a lymphatic-specific target or marker, particularly in liver research and pathology studies. Experimental designs should account for this potential dual vascular expression when interpreting results, especially in hepatic tissues .

How can researchers distinguish between LYVE1 expression in lymphatic versus blood vascular endothelium?

To accurately differentiate between LYVE1 expression in lymphatic and blood vascular endothelium, researchers should implement a multi-parameter approach:

• Co-staining with additional markers:

  • Lymphatic markers: Podoplanin, PROX1, VEGFR-3

  • Blood vessel markers: CD31 (higher expression in blood vessels), von Willebrand factor

• Morphological assessment:

  • Lymphatic vessels typically have irregular lumens and lack a complete basement membrane

  • Blood vessels have more regular lumens and pericyte coverage

• Tissue-specific considerations:

  • In liver tissue, expect LYVE1 expression in both lymphatics and sinusoids

  • In other tissues, expression is predominantly lymphatic

This combinatorial approach provides more reliable identification than using LYVE1 alone, especially in hepatic tissues where dual expression occurs .

What are the primary molecular functions of LYVE1, and how does the 25-235 a.a. fragment contribute to these functions?

LYVE1 serves multiple molecular functions in lymphatic biology, with the 25-235 a.a. fragment encompassing the critical functional domain. These functions include:

• Hyaluronan (HA) transport and turnover:

  • Mediates uptake of HA for catabolism within lymphatic endothelial cells

  • Facilitates HA delivery into lymphatic vessel lumens for transport to lymph nodes

  • The 25-235 a.a. region contains the complete HA-binding Link module essential for this function

• Surface localization of HA:

  • Sequesters HA on the surface of lymphatic endothelial cells

  • Creates an HA-rich microenvironment that influences cell migration and tissue homeostasis

• Signaling:

  • Participates in lymphatic-specific signaling pathways

  • Interacts with growth factors including PDGFB

The 25-235 a.a. fragment retains the full HA-binding capacity and can be used experimentally to study these interactions without the complications of membrane insertion that the full-length protein requires .

How does LYVE1 contribute to tumor metastasis, and what experimental evidence supports this role?

LYVE1 appears to play several roles in tumor metastasis, though the complete mechanisms remain under investigation:

• Lymphatic invasion pathway:

  • As a predominant lymphatic marker, LYVE1-positive vessels provide a conduit for tumor cell dissemination

  • Interaction between tumor-derived HA and lymphatic LYVE1 may facilitate tumor cell adhesion and entry into lymphatics

• Altered expression in tumors:

  • LYVE1 is absent from angiogenic blood vessels in human liver tumors

  • This differential expression may contribute to altered vascular permeability and function in tumors

• Potential roles in immune cell trafficking:

  • LYVE1-HA interactions modulate dendritic cell and macrophage migration

  • This may influence tumor-associated inflammation and immunity

Experimental evidence has largely come from studies using LYVE1 as a marker for tumor lymphangiogenesis. Interestingly, LYVE1 knockout models have not shown dramatic phenotypes, suggesting possible compensatory mechanisms or context-dependent functionality in metastasis .

What proteolytic processing affects LYVE1, and how does this impact its function?

LYVE1 undergoes significant proteolytic processing that affects its distribution and function:

• MT1-MMP (Membrane Type 1-Matrix Metalloproteinase) cleavage:

  • Cleaves LYVE1 at two specific sites: G64-L within the HA-binding domain and A235-L in the membrane proximal domain

  • Releases fragments of approximately 20, 30, and 50 kDa into the extracellular space

• Functional consequences of cleavage:

  • Shedding of the functional ectodomain (containing the 25-235 a.a. region)

  • Reduction of cell-surface HA-binding capacity

  • Potentially modulating lymphatic vessel functions including permeability and transport

• Regulation of cleavage:

  • Inhibited by MT1-MMP inhibitors like GM6001 and EDTA

  • May be induced by inflammatory stimuli or during tissue remodeling

This proteolytic processing represents a key regulatory mechanism for LYVE1 function and may explain why soluble forms of LYVE1 can be detected in biological fluids .

How do mutations at the MT1-MMP cleavage sites affect LYVE1 processing and function?

Mutations at the MT1-MMP cleavage sites of LYVE1 significantly alter its processing and potentially its biological function:

These experimental findings demonstrate that site-directed mutagenesis of the LYVE1 cleavage sites can be used to engineer cleavage-resistant variants for research or therapeutic applications. Such variants would maintain extended surface expression and potentially enhanced HA-binding capacity, allowing researchers to assess the biological significance of LYVE1 shedding in various experimental contexts .

What are the optimal expression systems for producing recombinant LYVE1 Human 25-235 a.a., and what yields can be expected?

Various expression systems have been successfully employed for producing recombinant LYVE1 Human 25-235 a.a., each with distinct advantages:

• Insect cell expression system:

  • High Five insect cells provide high-yield expression with proper folding

  • Typical yields: 1-5 mg/L of culture

  • Advantages: Proper glycosylation patterns closer to mammalian cells

  • Purification: Conventional chromatography techniques with His-tag affinity

• Mammalian expression systems:

  • COS-1 or HEK293 cells for transient or stable expression

  • Yields: 0.5-2 mg/L for transient systems

  • Advantages: Native glycosylation and folding

  • Often expressed as fusion proteins (e.g., with IgFc) to facilitate purification

• E. coli expression:

  • Generally less suitable for the functional domain due to improper folding

  • May be useful for linear epitope studies or structural analyses after refolding

The choice of expression system should be guided by the intended experimental application. For functional studies requiring proper folding and glycosylation, insect or mammalian systems are preferred, while bacterial systems may be suitable for structural studies after appropriate refolding protocols .

What are the key considerations for designing binding assays using LYVE1 Human 25-235 a.a.?

Designing effective binding assays with LYVE1 Human 25-235 a.a. requires careful attention to several technical parameters:

• Buffer conditions:

  • Optimal pH range: 7.0-7.5

  • Presence of divalent cations (Ca²⁺, Mg²⁺) can enhance binding

  • Inclusion of 0.05-0.1% BSA to reduce non-specific interactions

• HA preparation considerations:

  • Size-defined HA fragments yield more reproducible results

  • Biotinylated HA provides versatility for detection methods

  • High and low molecular weight HA may show different binding characteristics

• Plate-based binding assays:

  • Immobilization strategies: direct coating vs. capture antibody approach

  • Concentration ranges: typically 0.1-10 μg/ml of recombinant LYVE1

  • Detection methods: direct labeling vs. antibody-based detection

• Solution-based assays:

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Microscale thermophoresis for solution-phase interactions

  • Fluorescence polarization for studying smaller HA fragments

• Controls:

  • CD44 as a positive control for HA binding

  • G64A mutant as a reduced-function control

  • Pre-blocking with unlabeled HA to demonstrate specificity

These considerations help ensure reproducible and physiologically relevant binding data when working with the LYVE1 extracellular domain .

What mass spectrometry approaches are most effective for characterizing LYVE1 and its proteolytic fragments?

Multiple mass spectrometry (MS) approaches have proven effective for characterizing LYVE1 and its fragments, each with specific applications:

• MALDI-TOF MS:

  • Most suitable for confirming the molecular mass of intact recombinant LYVE1 (25-235 a.a.)

  • Can verify the 24.8 kDa expected mass of the purified protein

  • Provides rapid quality control for recombinant protein production

• LC-MS/MS (Tandem MS) for proteolytic fragment analysis:

  • Essential for identifying precise cleavage sites (e.g., G64-L and A235-L by MT1-MMP)

  • Can detect post-translational modifications including glycosylation sites

  • Enables mapping of the complete protein sequence through tryptic digestion

• Top-down proteomics:

  • Analysis of intact protein and larger fragments

  • Provides information on proteoforms and their relative abundance

  • Useful for characterizing complex mixtures of LYVE1 fragments

• Cross-linking MS:

  • Valuable for studying LYVE1 homodimerization and protein-protein interactions

  • Can reveal structural information about binding interfaces

For studying MT1-MMP-generated fragments specifically, synthetic LYVE1 polypeptides (such as 55L-75S and 225E-249R) can be digested with recombinant catalytic domain of MT1-MMP and analyzed by MS to confirm cleavage sites with high precision .

How can researchers effectively differentiate between membrane-bound and soluble forms of LYVE1 in biological samples?

Effectively distinguishing between membrane-bound and soluble LYVE1 in biological samples requires a combination of analytical approaches:

• Differential centrifugation and western blotting:

  • Sequential centrifugation separates membrane fractions (100,000-200,000g pellet) from soluble proteins

  • Western blotting with antibodies targeting different LYVE1 epitopes

  • Expected patterns: full-length (60-70 kDa) in membrane fractions vs. smaller fragments (20-50 kDa) in soluble fractions

• Epitope-specific detection:

  • Antibodies targeting N-terminal regions detect both membrane-bound and soluble forms

  • C-terminal-specific antibodies detect only intact membrane-bound forms

  • Using both antibody types enables differentiation between forms

• Glycosylation analysis:

  • Membrane-bound LYVE1 typically has complete glycosylation

  • Soluble forms may have altered glycosylation patterns

  • PNGase F or similar glycosidase treatment followed by western blotting reveals core protein sizes

• Flow cytometry for cellular samples:

  • Surface staining (non-permeabilized) detects membrane-bound LYVE1

  • Combined surface and intracellular staining distinguishes between locations

  • Can be combined with lymphatic endothelial markers for tissue analysis

These methods can be particularly important when studying MT1-MMP-mediated shedding of LYVE1, where both membrane-bound and soluble forms coexist in biological systems .

How can researchers establish an in vitro model to study LYVE1-mediated hyaluronan uptake and transport?

Establishing an effective in vitro model for LYVE1-mediated hyaluronan uptake and transport requires several components:

• Cell system selection:

  • Primary lymphatic endothelial cells (LECs) for physiological relevance

  • Stable LYVE1-expressing cell lines (e.g., LYVE1-transfected HEK293 or HMEC-1)

  • Comparison between wild-type and LYVE1-knockout cells to confirm specificity

• Labeled hyaluronan preparation:

  • Fluorescent labeling: FITC, Alexa Fluor, or rhodamine-conjugated HA

  • Size-defined HA fragments (optimal: 40-300 kDa range)

  • Biotinylated HA for non-fluorescent detection methods

• Uptake assay design:

  • Time course: Typically 5 minutes to 24 hours to capture both binding and internalization phases

  • Temperature conditions: 4°C (binding only) vs. 37°C (binding and internalization)

  • Quantification methods: Flow cytometry, confocal microscopy, plate reader

• Transport model options:

  • Transwell systems with LECs on porous membranes

  • Microfluidic devices with defined flow parameters

  • 3D lymphatic vessel organoids in extracellular matrix

• Validation controls:

  • LYVE1-blocking antibodies to confirm specificity

  • Competitive inhibition with excess unlabeled HA

  • Comparison with CD44-mediated uptake

• Analysis of internalization mechanisms:

  • Endocytic pathway inhibitors (e.g., dynasore, chlorpromazine)

  • Colocalization with endosomal/lysosomal markers

  • Comparison of internalization rates with HA degradation

This experimental system enables quantitative assessment of both HA binding and the subsequent internalization and transport processes mediated by LYVE1 .

What approaches can be used to study the interaction between LYVE1 and MT1-MMP in regulating lymphatic vessel function?

Several complementary approaches can be employed to investigate the regulatory interaction between LYVE1 and MT1-MMP in lymphatic vessel function:

• Cell culture models:

  • Co-culture of lymphatic endothelial cells (LECs) with MT1-MMP-expressing cells

  • LECs with inducible MT1-MMP expression systems

  • CRISPR/Cas9-mediated generation of LYVE1 cleavage-resistant mutants (G64A, A235V)

• Protein interaction analysis:

  • Co-immunoprecipitation of LYVE1 with MT1-MMP

  • Proximity ligation assays to visualize interactions in situ

  • FRET/BRET approaches for dynamic interaction monitoring

• Functional readouts:

  • HA binding capacity before and after MT1-MMP exposure

  • Lymphatic permeability assays using transendothelial electrical resistance

  • Tube formation assays to assess morphogenic impacts

• In vivo approaches:

  • Endothelial-specific MT1-MMP knockout mice (ΔEC mice)

  • Analysis of LYVE1 levels in lymphatic vessels from multiple organs

  • Tissue-specific inducible expression of wild-type or catalytic-dead MT1-MMP

• Inhibitor studies:

  • MT1-MMP inhibitors (GM6001, EDTA) and their effects on LYVE1 shedding

  • Time-course and dose-response analyses

  • Comparison with other metalloproteinases

This multifaceted approach enables comprehensive characterization of how MT1-MMP-mediated cleavage regulates LYVE1 availability and function in lymphatic vessels under both physiological and pathological conditions .

How does LYVE1 expression change in tumor-associated lymphatics, and what are the implications for cancer research?

LYVE1 expression undergoes significant changes in tumor-associated lymphatics with important implications for cancer research:

• Expression pattern alterations:

  • Upregulation in tumor-associated lymphangiogenesis

  • Heterogeneous expression levels within the same tumor

  • Absent from angiogenic blood vessels in human liver tumors

  • Reduced in pre-existing lymphatics adjacent to tumors

• Correlation with lymphatic metastasis:

  • Higher LYVE1⁺ vessel density often correlates with increased lymph node metastasis

  • The pattern of LYVE1 expression may predict metastatic routes

  • Differential LYVE1 expression between intratumoral and peritumoral lymphatics

• Mechanistic implications:

  • MT1-MMP-mediated LYVE1 shedding may alter the tumor microenvironment

  • Changes in HA binding and transport affect tumor interstitial fluid pressure

  • Potential role in modulating immune cell trafficking to lymph nodes

• Research applications:

  • LYVE1 as a marker for lymphatic vessel density quantification

  • Target for lymphatic-specific drug delivery

  • Potential biomarker in liquid biopsies (shed LYVE1 ectodomain)

These findings underscore the importance of considering both the presence and absence of LYVE1 in different vascular compartments when studying tumor biology. The differential expression in hepatic tissues is particularly noteworthy, as LYVE1 is present in normal liver sinusoids but absent in tumor-associated blood vessels .

What is the experimental evidence for LYVE1's dual expression in lymphatic and blood vascular endothelium in specific tissues?

The dual expression of LYVE1 in both lymphatic and blood vascular endothelium, particularly in hepatic tissues, is supported by several lines of experimental evidence:

• Immunohistochemical studies:

  • Co-localization of LYVE1 with blood vessel markers in normal liver sinusoids

  • Simultaneous detection in both vessel types using confocal microscopy

  • Differential staining intensity between lymphatic and blood sinusoidal endothelium

• Flow cytometry analysis:

  • Isolation of LYVE1⁺ endothelial cells from both lymphatic and hepatic vascular beds

  • Quantitative assessment of expression levels between different endothelial populations

  • Co-expression with other endothelial markers

• In situ hybridization:

  • Detection of LYVE1 mRNA in both vessel types

  • Confirmation that expression occurs at the transcriptional level

• Electron microscopy:

  • Ultrastructural localization of LYVE1 in liver sinusoidal endothelial cells

  • Distinct patterns of distribution between lymphatic and blood vessel endothelium

• Tissue-specific expression patterns:

  • Present in normal hepatic blood sinusoidal endothelial cells

  • Absent from angiogenic blood vessels in human liver tumors

  • Weakly present in microcirculation of regenerative hepatic nodules in cirrhosis

This evidence challenges the use of LYVE1 as an exclusive lymphatic marker, particularly in liver research, and necessitates careful interpretation when using LYVE1 for vessel identification in different tissue contexts .